Microalgae are versatile organisms capable of converting CO2, H2O, and sunlight into fuel and chemicals for domestic and industrial consumption. Thus, genetic modifications of microalgae for ...enhancing photosynthetic productivity, and biomass and bio-products generation are crucial for both academic and industrial applications. However, targeted mutagenesis in microalgae with CRISPR-Cas9 is limited. Here we report, a one-step transformation of Chlamydomonas reinhardtii by the DNA-free CRISPR-Cas9 method rather than plasmids that encode Cas9 and guide RNAs. Outcome was the sequential CpFTSY and ZEP two-gene knockout and the generation of a strain constitutively producing zeaxanthin and showing improved photosynthetic productivity.
Abstract
Microalgae can accumulate various carbon-neutral products, but their real-world applications are hindered by their CO
2
susceptibility. Herein, the transcriptomic changes in a model ...microalga,
Chlamydomonas reinhardtii
, in a high-CO
2
milieu (20%) are evaluated. The primary toxicity mechanism consists of aberrantly low expression of plasma membrane H
+
-ATPases (PMAs) accompanied by intracellular acidification. Our results demonstrate that the expression of a universally expressible PMA in wild-type strains makes them capable of not only thriving in acidity levels that they usually cannot survive but also exhibiting 3.2-fold increased photoautotrophic production against high CO
2
via maintenance of a higher cytoplasmic pH. A proof-of-concept experiment involving cultivation with toxic flue gas (13 vol% CO
2
, 20 ppm NO
X
, and 32 ppm SO
X
) shows that the production of CO
2
-based bioproducts by the strain is doubled compared with that by the wild-type, implying that this strategy potentially enables the microalgal valorization of CO
2
in industrial exhaust.
•AGP locus in Tetraslemis sp. was mutated by CRISPR-Cas9 RNP.•AGP mutants showed enhanced lipid production and the absence of starch synthesis.•Fatty acids in AGP mutants increased by 274% and 314% ...compared to that in wild type.•AGP mutants showed 2.3- and 2.7-fold higher lipid productivity than the wild type.
A marine green microalga, Tetraselmis sp., has been studied for the production of biomass and lipids in seawater culture. Since carbohydrate and lipid biosynthesis are competitive metabolic pathways, we attempted to increase lipid synthesis in Tetraselmis by inhibiting carbohydrate synthesis. The main regulatory enzyme in the starch synthesis pathway is ADP-glucose pyrophosphorylase (AGP). AGP loss-of-function mutants were developed using the CRISPR-Cas9 ribonucleoprotein (RNP) delivery system. AGP mutants showed a slight decrease in growth. However, the lipid content in two AGP mutants was significantly enhanced by 2.7 and 3.1 fold (21.1% and 24.1% of DCW), respectively, compared to that in the wild type (7.68% of DCW) under nitrogen starvation. This study is an example of metabolic engineering by genetic editing using the CRISPR-Cas9 RNP method in marine green microalgae. Consequently, starchless Tetraselmis mutants might be considered potential producers of lipids in seawater cultures.
Most sexual organisms inherit organelles from one parent, commonly by excluding organelles from the smaller gametes. However, post-mating elimination of organelles derived from one gamete ensures ...uniparental inheritance, where the underlying mechanisms to distinguish organelles by their origin remain obscure. Mating in Chlamydomonas reinhardtii combines isomorphic plus and minus gametes, but chloroplast DNA from minus gametes is selectively degraded in zygotes. Here, we identify OTU2p (otubain protein 2), encoded in the plus mating-type locus MT+, as the protector of plus chloroplast. Otu2p is an otubain-like deubiquitinase, which prevents proteasome-mediated degradation of the preprotein translocase of the outer chloroplast membrane (TOC) during gametogenesis. Using OTU2p-knockouts and proteasome inhibitor treatment, we successfully redirect selective DNA degradation in chloroplasts with reduced TOC levels regardless of mating type, demonstrating that plus-specific Otu2p establishes uniparental chloroplast DNA inheritance. Our work documents that a sex-linked organelle quality control mechanism drives the uniparental organelle inheritance without dimorphic gametes.
is being transformed from a model organism to an industrial organism for the production of pigments, fatty acids, and pharmaceuticals. Genetic modification has been used to increase the economic ...value of
. However, low gene-editing efficiency and position-effects hinder the genetic improvement of this microorganism. Recently, site-specific double-stranded DNA cleavage using CRISPR-Cas9 system has been applied to regulate a metabolic pathway in
. In this study, we proved that site-specific gene expression can be induced by CRISPR-Cas9-mediated double-strand cleavage and non-homologous end joining (NHEJ) mechanism. The CRISPR-Cas9-mediated knock-in method was adopted to improve gene-editing efficiency and express the reporter gene on the intended site. Knock-in was performed using a combination of ribonucleoprotein (RNP) complex and DNA fragment (antibiotics resistance gene). Gene-editing efficiency was improved via optimization of a component of RNP complex. We found that when the gene
was targeted, the efficiency of obtaining the desired mutant by the knock-in method combined with antibiotic resistance was nearly 37%; 2.5 times higher than the previous reports. Additionally, insertion of a long DNA fragment (3.2 and 6.4 kb) and site-specific gene expression were analyzed. We demonstrated the knock-out phenotype of
and on-site inserted gene expression of luciferase and mVenus at the same time. This result showed that CRISPR-Cas9-mediated knock-in can be used to express the gene of interest avoiding position-effects in
. This report could provide a new perspective to the use of gene-editing. Furthermore, the technical improvements in genetic modification may accelerate the commercialization of
.
To date,
is the most used species of microalgae in the food and feed additive industries, and also considered as a feasible cell factory for bioproducts. However, the lack of an efficient genetic ...engineering tool makes it difficult to improve the physiological characteristics of this species. Therefore, the development of new strategic approaches such as genome editing is trying to overcome this hurdle in many research groups. In this study, the possibility of editing the genome of
UTEX395 using clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) has been proven to target nitrate reductase (
) and adenine phosphoribosyltransferase (
). Genome-edited mutants,
and
, were generated by a DNA-mediated and/or ribonucleoprotein (RNP)-mediated CRISPR-Cas9 system, and isolated based on the negative selection against potassium chlorate or 2-fluoroadenine in place of antibiotics. The null mutation of edited genes was demonstrated by the expression level of the correspondent proteins or the mutation of transcripts, and through growth analysis under specific nutrient conditions. In conclusion, this study offers relevant empirical evidence of the possibility of genome editing in
UTEX395 by CRISPR-Cas9 and the practical methods. Additionally, among the generated mutants,
can provide an easier screening strategy during DNA transformation than the use of antibiotics owing to their auxotrophic characteristics. These results will be a cornerstone for further advancement of the genetics of
.
The Chlamydomonas reinhardtii truncated light-harvesting antenna 4 (tla4) DNA transposon mutant has a pale green phenotype, a lower chlorophyll (Chl) per cell and a higher Chl a/b ratio in comparison ...with the wild type. It required a higher light intensity for the saturation of photosynthesis and displayed a greater per chlorophyll light-saturated rate of oxygen evolution than the wild type. The Chl antenna size of the photosystems in the tla4 mutant was only about 65% of that measured in the wild type. Molecular genetic analysis revealed that a single plasmid DNA insertion disrupted two genes on chromosome 11 of the mutant. A complementation study identified the “chloroplast signal recognition particle 54” gene (CpSRP54), as the lesion causing the tla4 phenotype. Disruption of this gene resulted in partial failure to assemble and, therefore, lower levels of light-harvesting Chl-binding proteins in the C. reinhardtii thylakoids. A comparative in silico 3-D structure-modeling analysis revealed that the M-domain of the CpSRP54 of C. reinhardtii possesses a more extended finger loop structure, due to different amino acid composition, as compared to that of the Arabidopsis CpSRP54. The work demonstrated that CpSRP54 deletion in microalgae can serve to generate tla mutants with a markedly smaller photosystem Chl antenna size, improved solar energy conversion efficiency, and photosynthetic productivity in high-density cultures under bright sunlight conditions.
•The tla4 mutant of Chlamydomonas reinhardtii possesses a truncated light-harvesting antenna.•Disruption of the signal recognition particle 54 (CpSRP54) gene caused this phenotype.•3D modeling showed a difference in the Chlamydomonas and Arabidopsis CpSRP54 proteins.
Under high light, oxygenic photosynthetic organisms avoid photodamage by thermally dissipating absorbed energy, which is called nonphotochemical quenching. In green algae, a chlorophyll and ...carotenoid-binding protein, light-harvesting complex stress-related (LHCSR3), detects excess energy via a pH drop and serves as a quenching site. Using a combined in vivo and in vitro approach, we investigated quenching within LHCSR3 from
In vitro two distinct quenching processes, individually controlled by pH and zeaxanthin, were identified within LHCSR3. The pH-dependent quenching was removed within a mutant LHCSR3 that lacks the residues that are protonated to sense the pH drop. Observation of quenching in zeaxanthin-enriched LHCSR3 even at neutral pH demonstrated zeaxanthin-dependent quenching, which also occurs in other light-harvesting complexes. Either pH- or zeaxanthin-dependent quenching prevented the formation of damaging reactive oxygen species, and thus the two quenching processes may together provide different induction and recovery kinetics for photoprotection in a changing environment.
Zeaxanthin, a major xanthophyll pigment, has a significant role as a retinal pigment and antioxidant. Because zeaxanthin helps to prevent age-related macular degeneration, its commercial use in ...personalized nutritional and pharmaceutical applications has expanded. To meet the quantitative requirements for personalized treatment and pharmaceutical applications, it is necessary to produce highly purified zeaxanthin. In this study, to meet the quantitative requirements for industrial applications, we generated a double knockout mutant which is gene-edited by the CRISPR-Cas9 ribonucleoprotein-mediated knock-in system. The lycopene epsilon cyclase (LCYE) was edited to the elimination of alpha-branch of xanthophyll biosynthesis in a knockout mutant of the zeaxanthin epoxidase gene (ZEP). The double knockout mutant (dzl) had a 60% higher zeaxanthin yield (5.24 mg L.sup.- 1) and content (7.28 mg g.sup.- 1) than that of the parental line after 3 days of cultivation. Furthermore, medium optimization improved the 3-day yield of zeaxanthin from the dzl mutant to 6.84 mg L.sup.- 1. A Chlamydomonas strain with the elimination of lutein production by gene editing using CRISPR-Cas9 has been successfully developed. This research presents a solution to overcome the difficulties of the downstream-process for the production of high-purity zeaxanthin.
Photosynthetic organisms are exposed to various environmental sources of oxidative stress. Land plants have diverse mechanisms to withstand oxidative stress, but how microalgae do so remains unclear. ...Here, we characterized the Chlamydomonas reinhardtii basic leucine zipper (bZIP) transcription factor BLZ8, which is highly induced by oxidative stress. Oxidative stress tolerance increased with increasing BLZ8 expression levels. BLZ8 regulated the expression of genes likely involved in the carbon-concentrating mechanism (CCM): HIGH-LIGHT ACTIVATED 3 (HLA3), CARBONIC ANHYDRASE 7 (CAH7), and CARBONIC ANHYDRASE 8 (CAH8). BLZ8 expression increased the photosynthetic affinity for inorganic carbon under alkaline stress conditions, suggesting that BLZ8 induces the CCM. BLZ8 expression also increased the photosynthetic linear electron transfer rate, reducing the excitation pressure of the photosynthetic electron transport chain and in turn suppressing reactive oxygen species (ROS) production under oxidative stress conditions. A carbonic anhydrase inhibitor, ethoxzolamide, abolished the enhanced tolerance to alkaline stress conferred by BLZ8 overexpression. BLZ8 directly regulated the expression of the three target genes and required bZIP2 as a dimerization partner in activating CAH8 and HLA3. Our results suggest that a CCM-mediated increase in the CO2 supply for photosynthesis is critical to minimize oxidative damage in microalgae, since slow gas diffusion in aqueous environments limits CO2 availability for photosynthesis, which can trigger ROS formation.
PDF
